Clinicopathologic study of mantle cell lymphoma with epstein-barr virus infection: A case series and literature review

Background Mantle cell lymphoma (MCL) with Epstein–Barr virus (EBV) infection is rarely reported. The objective of this study was to analyze the prevalence and clinicopathological features of MCL with EBV infection in the largest series thus far. Methods After screening 138 cases of MCL, we identified eight cases of MCL with EBV infection. Results Most of them (7/8) had non-neoplastic bystander cells with positivity for EBV and no expression of latent membrane protein 1 (LMP1) and EBV nuclear antigen 2 (EBNA2). The cases of MCL with EBER positivity did not have abnormal immune function or other lymphomas. Moreover, their histopathological morphology was indicative of classical MCL. Cases of MCL with EBER positivity exhibited statistically significant differences in lactate dehydrogenase, anemia status, and MCL international prognostic index grouping (P=0.008, P=0.02, P=0.001, and P=0.011, respectively). The differences between the two groups in age, sex ratio, clinical manifestations, and immunohistochemical phenotypes were not statistically significant. Conclusions The incidence of MCL with EBV infection was low (5.8%). Clinicopathologically, cases of MCL with EBER positivity were similar to their EBV-negative counterparts. Our findings revealed that most cells infected by EBV in MCL are background cells rather than tumor cells. This is inconsistent with data from previous studies, indicating that tumor cells in MCL may not be prone to EBV infection.


Introduction
Epstein-Barr virus (EBV), also termed human herpes virus 4, exhibits an infection rate >90% worldwide (1). Etiologically, EBV is linked to lymphoproliferative lesions and malignant lymphomas of B-, T-, and natural killer-cell origin, manifesting in the form of asymptomatic latent infection in the vast majority of individuals (2,3). Some EBV infections are events that persist in the B lymphoid system, while others are the result of viral entry into T cells or natural killer cells (3). EBV infection is associated with disease progression, and patients with EBV-positive lymphoma, diffuse large B-cell lymphoma, have a poor prognosis (4). Recent studies have found that some mature small B-cell lymphomas, such as follicular lymphoma (FL) and marginal zone lymphoma (MZL), are associated with EBV infection (5)(6)(7)(8). Most of these cases are linked to aging, immunodeficiency, immunosuppression, and transplantation.
At present, there are few studies on EBV-associated mantle cell lymphoma (MCL), and most of them are case reports (9)(10)(11)(12)(13)(14)(15)(16). These studies have established the connection between EBV and MCL. However, due to the complex life cycle and biological behavior of EBV, its role in MCL remains unknown. According to the currently available research studies, it is hypothesized that EBV may be associated with the transformation of MCL to Hodgkin's lymphoma (HL) or more aggressive lymphoma (16). Therefore, it is necessary to correctly recognize and diagnose MCL with EBV infection.
In this study, we analyzed eight MCL cases with EBV infection, including their clinical manifestations and laboratory findings, to investigate whether MCL with EBV infection has unique clinicopathological features.

Clinical and pathological characteristics assessment
Clinical data, including age, sex, biopsy site, systemic lymphadenopathy, extra-lymph node lesions, bone marrow involvement, B symptoms, Ann Arbor stage, and MCL international prognostic index (MIPI) groups, were analyzed. Additionally, laboratory examinations, including red blood cell (RBC) count, hemoglobin count, lactate dehydrogenase (LDH), leukocyte count, as well as the absolute value of monocytes and beta-2-microglobulin (b2-MG), were analyzed.
In situ hybridization (ISH) for EBV and CD79a/CD34 immunohistochemical double labeling EBV detection was performed by ISH using EBV-encoded RNA (EBER) probes (Zhongshanjinqiao Biotechnology Co., Ltd.) (Beijing, China) according to the experimental protocol provided by the manufacturer. This technique can determine the type of cells infected by EBV. The positive signal of EBER was located in the nucleus and was shown with a brownish yellow/ red. The positive signal of CD79a/CD3 was located on the cell membrane of B/T lymphocytes. Therefore, only tumor cells that exhibit positivity both in the nucleus (EBER) and membrane (CD79a) are judged to be EBV-positive tumor cells. Subsequently, specimens were observed using a microscope (Olympus, Japan).

Statistical analysis
Data are presented as the mean ± standard deviation, range, frequencies (number of cases), and percentages as appropriate. Student's t-test using two independent samples was employed for comparisons between groups; the median and quartile interval were used to determine data skewness, and the Mann-Whitney U test was used for comparison between groups. Statistical analyses were performed using the SPSS software (version 17.0; IBM Corp, Armonk, NY, USA). P-values < 0.05 denoted statistically significant differences.

Results
Clinical features of EBV-positive and EBV-negative MCL patients Screening of 138 cases of MCL revealed eight EBER-positive cases. Therefore, the infection rates with and without EBV in MCL were 5.8% (8/138) and 94.2% (130/138), respectively. Due to the lack of complete clinical data and pathological tissue, the data of 52 patients were excluded. Therefore, 8 EBER positive MCL patients and 78 EBER negative MCL patients were included. Clinical characteristics of MCL patients with and without EBER positivity were analyzed and compared using the chi-squared test (Table 1). There were statistically significant differences in RBC count, hemoglobin count, LDH, and MIPI groups (P=0.008, P=0.02, P=0.001, and P=0.011, respectively). RBC and LDH increased in all MCL patients with EBER positivity, and the MIPI score indicated medium/high risk. There were no significant differences between MCL patients with and without EBER positivity for the mean age (P=0.342), age distribution (P=0.46), sex (P>0.999), systemic lymphadenopathy (P=0.343), extra-lymph node lesions (P=0.147), bone marrow involvement (P=0.195), B symptoms (P=0.588), Ann Arbor stage (P=0.585), and leukocyte count (P=0.558), as well as absolute value of monocytes (P=0.611) and b2-MG (P>0.999). Of the eight MCL patients with EBER positivity, six were males (75%) and two were females (25%), showing a slight male predominance (male:female=3:1). The median age was 66.5 years (range: 43-80 years); two patients were aged <60 years (25%); and six cases were aged ≥60 years (65%).
All patients had multiple lymph node enlargement; seven patients (87.5%) had extra-lymph node lesions, whereas one patient (12.5%) did not have extra-lymph node lesions. Four patients (50%) had bone marrow involvement, two patients (25%) did not exhibit bone marrow involvement, and bone marrow biopsy was not performed in two cases (25%). Hepatosplenomegaly was found in three cases (37.5%); the remaining five cases (62.5%) did not have hepatosplenomegaly. All patients had decreased RBC and hemoglobin counts; two cases (25%) had increased absolute value of monocytes; six cases (75%) had normal absolute value of monocytes; and one (12.5%) and seven patients (87.5%) had increased and normal white blood cell counts, respectively. Serum LDH levels were elevated in six cases (75%), and could not be evaluated in two cases (25%). b2-MG was elevated in three patients (37.5%), normal in one patient (12.5%), and not detected in four

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immunosuppression/immunodeficiency or B symptoms. Two patients (25%) and four patients (50%) had Ann Arbor stages IIIA and IVA, respectively. The Ann Arbor stage could not be clearly assessed in two patients (25%) without bone marrow biopsy. According to the MIPI score, four (50%) and two (25%) patients were in the medium-and high-risk groups, respectively. It was not possible to detect LDH in two patients (25%); thus, these patients were not evaluated for MIPI. One patient was treated with CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone), three patients were treated with E-CHOP (CHOP and etoposide), and one patient was treated with CHOE (cyclophosphamide, adriamycin, vincristine, and etoposide); three patients were not treated with chemotherapy (Table 2).

Histological and immunophenotypic analysis of EBV-positive and EBVnegative MCL patients
The chi-squared test was used to analyze and compare the immunohistochemical findings of MCL patients with and without EBER positivity (Table 3). There was no significant difference in the expression of CD5 (P=0.641), SOX11 (P=0.17), CD10 (P>0.999), BCL-6 (P=0.396), and Ki-67 (P=0.707), with 30% as the threshold between MCL patients with and without EBV infection. However, the difference in CCND1 expression between the two groups was statistically significant (P<0.001). CCND1 expression was negative in part of MCL patients without EBV infection (5/78), but positive in MCL patients with EBV infection.
In the eight MCL patients with EBV infection, the morphology was indicative of classical MCL, the tumor cells were small-to-medium-sized lymphocytes, the cell morphology was single, the nucleus was slightly irregular, similar to the central cell, the nucleolus was not obvious, mitosis was rare, scattered vascular vitreous degeneration was observed, and tissue-like cells were observed in the tumor background; however, Hodgkin and Reed/Sternberg (HRS)-like cells were not observed (Figure 1, cases 1 and 4). CD20, CD79a, and CCND1 were diffusely expressed in all patients (100%) (Figures 2A, B, case 2). Six cases (75%) expressed CD5 ( Figure 2C, case 4), whereas two cases (25%) did not express. Three cases (37.5%) expressed SOX11 ( Figure 2D, case 3), whereas five cases (62.5%) did not. Seven cases (87.5%) expressed BCL-2, whereas one case (12.5%) did not. Three cases (37.5%) expressed BCL-6, whereas five cases (62.5%) did not. LMP1 ( Figure 2E, case 1) and EBNA2 ( Figure 2F, case 1) were not detected in any cases. Ki-67 ranged 5-80%; four cases (50%) were ≤30% and four cases (50%) were >30%. The results of immunohistochemistry and CCND/IgH-t (11;14) chromosome rearrangement and translocation in tissues of these eight cases are shown in Table 4.  In most cells, EBER and CD79a immunohistochemical double-label staining revealed that they were not expressed on A B D C FIGURE 1 Histopathological features of MCL with EBER positivity. In case 1, at low magnification (×200), the tumor cells were small-to-medium-sized lymphocytes, and the cell morphology was single (A). At high magnification (×400), the tumor nucleus was slightly irregular, similar to the central cells, the nucleolus was not obvious, mitosis was rare, and tissue-like cells were observed in the tumor background; however, HRS-like cells were not found (B). In case 4, at low magnification (×200), the tumor cells were small-to medium-sized lymphocytes, with scattered vitreous degeneration of blood vessels (C). At high magnification (×400), the nucleus of the tumor was slightly irregular, similar to the central cells, the nucleolus was not obvious, mitosis was rare, and there were no HRS-like cells in the background (D).   Case CD20 CD79a CD5 CyclinD1 SOX11 CD43 CD10 BCL-6 BCL-2 LMP-1 EBNA2 FISH t(11;14) Ki-67 This may indicate that EBV infection often occurs in patients with FL and MZL after transplantation or immunosuppressive therapy, or with normal immune function; subsequently, the condition transforms into higher-grade or more invasive lymphoma. Of note, there was no conversion to high-grade FL or DLBCL among the six cases of low-grade FL with EBV positivity in background cells reported by Mackrides et al. (6) This suggests that, even in the presence of EBV infection, the condition may not necessarily develop into a higher grade or more invasive lymphoma. This may be related to the fact that EBV does not infect tumor cells or the time of infection. According to Camacho Casta ñ EDA FI (8), one case of A B D C FIGURE 4 Immunohistochemical double-staining for EBER ISH and CD79a/CD3. A mixture of cell membrane-positive cells and nuclear-positive cells (A) (case 1). The cell membrane-positive cells were distributed in the tumor area, while most of the nuclear-positive cells were scattered in the inter-tumor area (B) (case 4). Most cases did not show membrane and nuclear positivity at the same time (magnification: ×400). EBER and CD3 immunohistochemical double-label staining revealed that EBER-positive cells were mainly distributed in the area of CD3-positive T cells (C) (case 4). A small number of EBER-positive cells could be clearly classified as CD3-positive cells (D) (case 4). Images (C, D) were captured at ×200 and ×400 magnification, respectively.
intranodal MZL was completely resolved after six cycles of chemotherapy. Lymph node biopsy showed scattered EBVpositive cells; 10 years later, the condition transformed into EBV-positive DLBCL. However, the article did not clearly state that the EBV-positive cells in the patient's first biopsy were tumor cells. In 2017, Ohashi et al. (21) found 29 patients with EBV-negative DLBCL and EBV-positive background cells among 664 patients with DLBCL. Their clinical manifestations were not significantly different from those of patients with EBVnegative DLBCL without EBV-positive background cells; nevertheless, their prognosis was poor. Among them, 33% (7/ 21) of patients expired within 2 years after diagnosis, and the patients with the longest survival time were followed up for 76 months, confirming the poor prognosis of these patients. This suggests that infection of background cells with EBV is a long early pathogenic process versus infection of tumor cells with this virus. In 1995, Hummel et al. (22) proposed that in case of EBV infection of a small number of tumors and paratumoral cells, the infection may occur after malignant transformation of the tumor. In the eight cases examined in this study, most EBVinfected cells were scattered near the tumor cells, EBER-positive cells gathered near the tumor cells in only one case, and EBERpositive cells were few. There was only one case with >100 cells, most cases had 10-100 cells, and one case had only one EBERpositive cell. Therefore, we hypothesized that infection of MCL patients with EBV may occur after the diagnosis of MCL.
We found that the immunophenotype of MCL with EBER positivity was similar to that of MCL without EBER positivity. Generally, patients with classical MCL express B cell markers, CD5, CCND1, and SOX11, and most exhibit t (11;14) chromosome rearrangement and translocation. Although the eight cases of MCL with EBV infection in this study demonstrated heterogeneous immunohistochemical expression, some cases did not express CD5 and SOX11, while other showed abnormal expression of BCL-6. However, these heterogeneities did not reveal statistically significant differences versus cases of MCL without EBV infection. In this study, eight cases expressed CD20, CD79a, and CCND1; of those, six cases expressed CD5. Some studies have found that patients with CD5-positive and CD5-negative MCL exhibit similar clinicopathological features to a large extent; however, patients with CD5-negative MCL have a better prognosis than those with CD5-positive MCL (23). Of the eight EBV-positive cases, five did not express SOX11, whereas three did. Studies have shown that SOX11-negative MCL is linked to a higher incidence of leukemia without lymph node involvement, classic histological morphology, and a lower Ki-67 index (24). In contrast, the clinical course of leukemic MCL without lymph node involvement is less aggressive than that of classical MCL. The BCL-6 gene encodes a transcriptional repressor protein that is mainly confined to germinal center B cells, while MCL is derived from pre-germinal center B cells or mantle B cells. In this study, five cases did not express BCL-6, whereas three cases did. Although abnormal BCL-6 expression has been rarely reported in MCL, it is associated with a high Ki-67 index and cytogenetic aberrations involving the BCL-6 gene (25,26). LMP1 and EBNA2 were not detected in the eight cases, indicating that they were not in the latent infection mode II or III (27). This finding was consistent with the results reported by Hummel et al. (28) in 1992.
The clinical features of the eight MCL patients with EBER positivity included in this study were similar to those of MCL patients without EBV infection. There were no significant differences in the age of onset, sex ratio, and most clinical manifestations. However, MCL patients with EBER positivity had elevated LDH, progressive anemia, and MIPI groups versus those without EBV infection. Most patients in this study were males and aged ≥60 years. Furthermore, most patients were in the late clinical stage (Ann Arbor stage III or IV), with multiple lymph node enlargement and anemia. Extranodal lesions were common (gastrointestinal tract and parotid gland). Approximately half of the patients had hepatosplenomegaly and bone marrow involvement; however, there were no B symptoms. Most patients had elevated LDH, and a few patients had elevated absolute values of b2-MG and monocytes. The increase of the first two indices indicated a poor prognosis (29, 30). In 2020, Zhou et al. (31) showed more EBV DNA-positive patients versus EBV DNA-negative patients in the group with high absolute monocytes. Ki-67 ≥30% in MCL is unfavorable to the overall survival of patients (32). Although Ki-67 was ≥30% in half of the patients analyzed in this study, the rate was similar to that observed in MCL patients without EBV infection. Follow-up analysis revealed that the 5-year overall survival rate of patients in the low-risk group was 60%, and the median survival time of patients in the medium-risk group was 51 months. The prognosis of patients in the high-risk group was poor, and the median survival time was only 29 months. Only six patients were evaluated for MIPI in this study, because two patients were not tested for LDH. All six cases were in the medium-and high-risk groups (four and two cases, respectively), suggesting that the prognosis of these patients may be slightly worse than that of other patients. Based on the follow-up of this study, the overall survival time of four patients was as short as 6 months and as long as 5 years.
In summary, the incidence of MCL with EBER positivity was low and mostly in background cells, in contrast to other mature small B-cell non-HL with EBER positivity ( Figure 5). In pathological diagnosis, pathomorphology, immunohistochemical staining, and clinical features cannot be used to evaluate the presence of EBV infection in MCL. This is because the clinicopathological features of MCL with EBV infection are similar to those of MCL without EBV infection. Patients with elevated LDH, progressive anemia status, and medium-or high-risk MIPI grouping are more likely to have EBV infection. Pathologists can detect EBV in patient tissue to determine the prognosis and disease progression. Additional research studies involving more samples and data are warranted to further clarify the clinicopathological features and biological significance of MCL with EBV infection.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
Studies were performed according to the Declaration of Helsinki. The procedures were reviewed and approved by the Ethics Committee of Chongqing University Cancer Hospital (CZLS2022065-A). The consent for publication was obtained from all participants for the publication of this study.